Fig 2: Protective effects of XXMD on LPS-induced acute lung injury in vivo. ALI model was induced by LPS intraperitoneal injection (10 mg/kg) with or without intragastric administration of XXMD (50 and 100 mg/kg), NAC (400 mg/kg), or DEX (2.5 mg/kg) 3 days after LPS treatment. (a) H&E, (b) IHC staining for NLRP3, (c, d) the expression levels of ZO-1, CLDN4, NLRP3, caspase 1, and (e) the expression levels of IL-1ß and IL-18 in bronchoalveolar lavage fluid were detected. Scale bar: 100 µm. Data are presented as the means ± SD, n = 6. ***P < 0.001 compared with control; ##P < 0.01 and ###P < 0.001 compared with LPS.

Fig 3: Effects of XXMD on ROS production, hyperpermeability, and NLRP3 inflammasome in HPAEpiC after NLRP3 overexpression. (a, b) HPAEpiC were transduced with pLVX-Puro-NLRP3, and the level of NLRP3 was detected. These cells were transduced with pLVX-Puro-NLRP3 or blank pLVX-Puro vector and then treated with XXMD (1 mg/mL) for 24 h and (c) the cell viability analysis, (d) TEER, (e) FITC permeability, (f, g) the expression level of ZO-1, CLDN4, NLRP3, and caspase 1, and (h) the levels of IL-1ß and IL-18 were determined. Data are presented as the means ± SD, n = 3. ***P < 0.001 compared with vector; ##P < 0.01 and ###P < 0.001 compared with NLRP3.

Fig 4: SARS-CoV-2-challenged hamster lungs faithfully recapitulate AT2 cytopathies in COVID-19. a. Schematic showing the experimental design for COVID-19 modeling in golden Syrian hamsters. Uninf, uninfected; Den3 and Anti-CoV-2 indicate SARS-CoV-2 challenged groups that received either a control mAb or the clone CC12.2 of anti-CoV-2 IgG,31 respectively. PFU, plaque-forming units. b. Heatmap showing transcripts/million (TPM)-normalized gene expression of specific genes associated with AT2 senescence (CDKN2A, TP53), damage associated transition state progenitor (DATP) arrested state (CLDN4, KRT8), ER stress (HSPA5/GRP78) and SASP-state (SERPINE1, COL1A, COL3A). c-d. Lungs harvested from the uninfected, infected, and infected but anti-CoV-2 IgG and infected but EIDD-2801 treated hamsters have been analyzed for the abundance of collagen deposition by Trichrome staining and quantified by ImageJ. Representative images are shown panel c. Scale bar = 200 µm. Results of the quantification are displayed as bar plot in D. Statistical significance was analyzed by one way ANOVA. Error bars represent S.E.M; n = 3. e-f. Lungs harvested from the uninfected, infected, and infected but anti-CoV-2 IgG groups were analyzed by IHC for the indicated proteins and quantified by ImageJ- IHC profiler. Representative images are shown panel E. Scale bar = 200 µm. Results of the quantification are displayed as violin plots in F. Statistical significance was analyzed by one way ANOVA; n = 4.

Fig 5: The role of NLRP3 overexpression in the protective effect of XXMD on LPS-induced ROS production, hyperpermeability, and NLRP3 inflammasome. HPAEpiC transduced with pLVX-Puro-NLRP3 or blank vector were treated with XXMD (1 mg/mL) for 3 h and then with LPS (10 mg/L) for 24 h, and the (a) TEER, (b) FITC permeability, (c, d) the expression of ZO-1, CLDN4, NLRP3, caspase 1, and (e) the levels of IL-1ß and IL-18 were determined. Data are presented as the means ± SD, n = 3. ***P < 0.001 compared with control; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with LPS; ???P < 0.001 compared with LPS + XXMD.